Helicobacter pylori is a curved gram-negative bacterium found in the upper gastrointestinal tract of humans. Since the first isolation of the bacterium in 1982, a huge amount of evidence has accumulated on the association of H. pylori with various gastric disorders, including dyspepsia (heartburn, bloating and nausea), symptomatic or asymptomatic inflammation of gastric mucosa manifested as chronic superficial gastritis or chronic active gastritis, peptic ulcers of the stomach and duodenum, and even gastric cancer and various gastric lymphomas [Dunn, B. E., et al., Clinical Microbiology Reviews 10 (1997) 720-740]. At present, it is believed that nearly all cases of peptic ulcers formerly thought to be idiopathic are actually caused by H. pylori infection [NIH Consensus Conference, JAMA 276 (1994) 1710].
H. pylori is a world wide human pathogen. The other known species carrying the bacterium is nonhuman primates. H. pylori infections have been connected to the socio-economic development: in developing countries 70 to 90% of the population carries the bacterium, whereas in developed countries the prevalence of the infection is approximately 25 to 50%. The infection is acquired in childhood, usually before the age of 10 years, and is believed that the rate of the incidence decreases with improved hygiene. However, the route of transmittance of the infection is not definitely known, although faecal-oral and oral-oral routes are thought to be most important (Dunn, B. E., et al., supra).
Various methods and assays, both invasive and non-invasive, are available for the diagnosis of H. pylori infection. The invasive methods involve gastric or duodenal biopsies. The biopsy samples can be examined visually or histologically, cultured for the bacteria, tested for the urease enzyme produced by H. pylori, or analysed with gene technology. Commercial products are available for most of these methods. Non-invasive methods include serological tests for the detection of antibodies to H. pylori and urea breath test using 13C or 14C-labelled urea, for both of which multiple commercial tests are available. Additionally, assays measuring substrate metabolism of H. pylori in serum [Moulton-Barret, R. G., et al., Am. J. Gastroenterol 88 (1993) 369-374] and in urine [Pathak, C. M., et al., Am. J. Gastroenterol 89 (1993) 734-738] have been described.
Immunoassays measure the presence of IgG, IgA or IgM antibodies against H. pylori in patients' serum or blood samples (see, for example, U.S. Pat. No. 5,262,156; Pyloriset EIA-A and EIA-G, Orion Diagnostica, Finland), urine samples (see, for example, U.S. Pat. No. 5,262,156), and saliva or other mucous secretion specimen (see, for example, U.S. Pat. No. 6,068,985; Home Helicobacter Test, Ani Biotech Oy, Finland). The determination of antibodies against H. pylori suffer from several drawbacks, such as the strong dependence of the antigen preparation which is used to capture the antibodies, cross reactions of antibodies from related bacterial species, and the relatively long time needed for reliable test results. The accuracy of the so-called “office-based” or “near-patient” tests offered for use in doctor's offices is poorer than that of conventional laboratory assays. [Cohen, H., et al., Gastroenterology 110 (1996) A83; Sadowski, D., et al., Gastroenterology 110 (1996) A246]. Importantly, these assays relying on the detection of specific antibodies against H. pylori are less suitable for use in the evaluation and follow-up of the treatment and cure, since the elevated antibody levels maintain for a long period of time after the treatment and cure of the infection. Follow-up studies show great variation in the decline of the antibody levels after treatment [Kosunen, T. U., et al., Lancet 339 (1992) 893-895; Cutler, A., et al., Dig. Dis. Sci. 38 (1993) 2262-2266], but usually several months are needed for a decline, which reliably predicts the cure.
The detection of H. pylori antigens or metabolites instead of specific antibodies against H. pylori in a biological sample addresses this drawback. U.S. Pat. Nos. 5,716,791, 5,871,942 and 5,932,430 disclose, inter alia, methods for the detection of H. pylori antigens in faecal samples by complexing the antigen with a polyclonal antibody and detecting the complex thus formed by a second antibody. International patent application WO01/44815 discloses the detection of H. pylori antigens in a blood samples with, for instance, an ELISA method. These methods are suggested for the follow-up of the effect of the treatment of H. pylori infection.
However, the conventional immunoassays rely on a marker molecule such as a radioactive label, an enzyme label, a fluorescent label or chemiluminescent label, and are laborious and time-consuming, since several incubation, washing and separation steps are needed before the actual detection. Additionally, for a reliable performance, they require a skilled personnel and rather expensive apparatuses. The sample, especially a faecal sample, may also represent a problem. Many patients find the collection of one faecal sample, let alone the collection of several faecal samples necessary for the follow-up, unpleasant and not hygienic, and their compliance to the treatment may suffer. Similarly, the personnel may dislike the handling of the faecal specimen and the preparation of samples for such assays due to the inherent infection risk.
Further improved diagnostic methods for the diagnosis of H. pylori infections are obviously needed.
Accordingly, one object of the present invention is to provide highly sensitive and specific methods and means for the non-invasive detection and determination of H. pylori antigens and/or metabolites produced by the bacterium in a biological sample.
Another object of the present invention is to provide improved methods and means for a reliable follow-up of the effect of pharmacotherapy in combating H. pylori infection and for the ascertainment of the cure of the patient with minor inconvenience to the patient.
A further object of the present invention is to provide improved methods and means for the detection of H. pylori infection, the methods being reliably applicable to the use in doctor's offices and in heath care centres, where the technical skills and routine of personnel may not be as advanced as in clinical laboratories.
A further object of the present invention is to provide improved methods and means for the detection of H. pylori infection, the methods being simple, rapid and real-timed so that the test results can be obtained even during the patient's visit at the hospital or the doctor's office, whereby several patient calls can be avoided.